Overview of AnemiaVol 1. 15th ed. New York, New York: McGraw-Hill; 2001:348-354 Some Etiologies of Anemia Physical •Trauma •Burns •Frostbite •Prosthetic valves and surfaces

Unrestricted © Siemens Healthcare Diagnostics Inc., 2017

Overview of Anemia:Diagnostic Testingin the Clinical &Laboratory MedicineMonet N. Sayegh, M.D.Physician Consultant

Monet N. Sayegh, M.D

. Page 2 | Unrestricted © Siemens Healthcare Diagnostics Inc., 2017A91LD-CAI-17878-S1-4A00

Anemia World Statistics

Global

• Iron Deficiency Anemia is the World’s most common micronutrient deficiency

• As many as 4-5 billion people (66-80% of population) may be iron deficient

• Categorized by WHO as one of the top ten most serious health problems in the modern world

• 1.62 billion people (>30% population) are anemic

• 818 million women worldwide (both pregnant and non-pregnant) and young children suffer from anemia and over half of these, approximately 520 million, live in Asia

• United States:~4% of men and 8% of women have values lower than normal values

Int J Gen Med. 2011; 4: 741–750.



Prevalence of Anemia by Age Group

0

10

20

30

40

50

60

0-4 Years 5-14 Years Nonpregnant Women Pregnant Women Men Elderly

Developing Countries

Industrialized Countries

McLean E, Public Health Nutrition, May 23:1-11,2008

Prevalence of Anemia by Age Group in Industrialized and Developing Countries, 1998

Per

cen

t



US Percent of Persons with Iron Deficiency

National Health and Nutrition Examination Survey 2003-2006

Children

Age 1 - 3 years

14%

Children

Age 3 - 5 years

4%

Females

Age 12 - 19 years

9%

Females

Age 20 - 49 years

9%



Anemia

DefinitionReduction in blood transport of oxygen due to a deficiency in red blood cells

• Anemia is defined by the WHO as a hemoglobin concentration of <12 g/dL for women and <13 g/dL for men..

Veng-Pedersen P, et. al. Pharm Res. Nov 2002;19(11):1630-5

Parameters

• Hematocrit – Percentage of blood volume as RBCs

• Hemoglobin – Concentration of hemoglobin in blood

• Mean Corpuscular Volume (MCV) – Average size of RBC

• Mean Corpuscular Hemoglobin (MCH) – Average hemoglobin content of RBC

• Mean Corpuscular Hemoglobin Concentration (MCHC) -The amount of hemoglobin relative to the size of the cell (hemoglobin concentration) per red blood cell

• RDW – Red cell distribution width

• Reticulocyte Hgb Content (CHr)

• %Hypo



1998-2014 Mayo Foundation for Medical Education and Researchhttp://www.mayoclinic.org/diseases-conditions/vitamin-deficiency-anemia/basics/risk-factors/con-20019550

Groups at High Risk

• Menstruating women

• Pregnant and breastfeeding women

• Babies, especially if premature

• Children going through puberty

• Vegetarians

• People with cancer, stomach ulcers and some chronic diseases (Renal Dialysis)

• GI surgery

• People on fad diets

• Athletes



Bone Marrow

Erythroid Stem Cell – Rubriblast - 1%

Reticulocyte

Metarubricyte 5 - 10%

Prorubricyte 1 - 4%

Rubricyte 10 - 20%

Red Cell Production (Erythropoiesis)

Hematology: Basic Principles and Practice. 5th ed. Philadelphia, PA: Churchill Livingstone; 2009:Chap 34:439-46.

Oxygen Sensor

Epo

Blood Vessel

Kidney

Effective ErythropoiesisRequires 20mg of Iron per day

AA, Vitamin B12, 6, Folic Acid, Ni, Co, EPO

Multi-potent Stem Cell

RBC survival~120 days

RNA Protein Synthesis

Mitochondria and Ribosomes

Peripheral blood

released from BM after 2-3 days and 1% circulate for additional 1-2 days

3 - 4 DAYS

EpomRNA



Increased RBC Destruction (Blood loss/hemolysis)

• Elevated reticulocyte count

Red cell maturation defects (ineffective erythropoiesis)

• Slight to moderately elevated reticulocyte count

• Macrocytic or microcytic anemia

Decreased RBC Production(hypoproliferation)

• Marrow production defects

• Low reticulocyte count

• Little or no change in red cell morphology (a normocytic, normochromic anemia)

Three Main Causes of Anemia



Nutritional

• Iron deficiency

• Vitamin B-12 deficiency

• Folate deficiency

• Starvation and generalized malnutrition

Infectious

• Viral - Hepatitis, infectious mononucleosis, cytomegalovirus

• Bacterial - Clostridia, gram-negative sepsis

• Protozoal - Malaria, leishmaniasis, toxoplasmosis

Adamson JW, et.al. Harrison's Principles of Internal Medicine. Vol 1. 15th ed. New York, New York: McGraw-Hill; 2001:348-354

Some Etiologies of Anemia

Physical

• Trauma

• Burns

• Frostbite

• Prosthetic valves and surfaces

Chronic disease and malignant

• Renal disease

• Hepatic disease

• Chronic infections

• Neoplasms

• Collagen vascular diseases



Some Genetic Causes of Anemia

• Hemoglobinopathies

• Thalassemia's

• Enzyme abnormalities of the glycolytic pathways

• Defects of the RBC cytoskeleton

• Congenital dyserythropoietic anemia

• Rh null disease

• Hereditary xerocytosis

• Abetalipoproteinemia

• Fanconi anemia


Unaffected“Carrier”

Father

Unaffected“Carrier”Mother

Unaffected1 in 4 chance

Unaffected “Carrier”2 in 4 chance

Affected1 in 4 chance



Red = In severe anemia

Eyes - Yellowing

Skin - Paleness- Coldness- Yellowing

Resiratory- Shortness of breath

Muscular- Weakness

Intestinal- Change stool color

Central - Fatgue- Dissiness- Fainting

Blood vessels- Low blood pressure

Heart- Palpitations- Rapid heart rate- Chest pain- Angina- Heart attack

Spleen- Enlargement

Symptoms of Anemia

Int J Gen Med. 2011; 4: 741–750. http://www.webnat.com/articles/Anemia.asp

Depending on the severity, the symptoms of anemia may include:

• Pale skin • Fatigue • Weakness • Tiring easily • Breathlessness • Orthostatic hypotension – this may happen

after acute blood loss, like a heavy period • Frequent headaches • Racing heart or palpitations • Becoming irritated easily • Concentration difficulties • Cracked or reddened tongue • Loss of appetite • Strange food cravings



Pulmonaryinsufficiency

Tissuehypoxia Shock

Complications of Severe Anemia


Themost serious

complications ofsevere anemia

arise from

Hypotension

Coronaryinsufficiency

Death



Initial Evaluation

History and Physical Exam

• Eating ice or clay (Pica)

• Dyspnea

• Chest pain

• Medications

• Conjunctival pallor

Laboratory Evaluation

• CBC with differential

• Peripheral smear

• Reticulocyte hemoglobin content

• Iron Studies (iron, TIBC, transferrin, ferritin)

• Vitamin B12 and Folate

• Evaluation for hemosiderinuria, hemoglobinuria, and pulmonary hemosiderosis

• Hemoglobin electrophoresis and measurement of hemoglobin A2 and fetal hemoglobin



Suggested Algorithm for the Diagnosis of Anemia

Hematology: Basic Principles and Practice. 5th ed. Philadelphia, PA: Churchill Livingstone; 2009:Chap 34:439-46.

MCV < 80 = Microcytic MCV 80-100 = Normocytic MCV > 100 = Macrocytic

Ferritin

Fe Deficient Fe Normal

IDA Anemia of Chronic Disease or

Hemoglobinopathy

Vitamin B12 & Folate

Normal Low

Reticulocyte Count

LowHemolysis or Blood LossACD ,CRF, Aplastic anemia , Protien energy malnutrition

or Marrow Failure

Obvious Cause Consider BM MDS, Liver Dz, Chemo

Myxedema

Establish Cause

Low Hb = AnemiaMCV

High



Daily Recommended Amount of Iron (mg) by Gender and Age

Int J Gen Med. 2011; 4: 741–750.

Age Amount (mg)

Adult

Males >18 years 8 mg

Females

> 50 Years 8 mg

19 - 50 Years 18 mg

Pregnant 27 mg

Breastfeeding 9 - 10 mg

Adolescent Males 9 - 18 Years 8 - 11 mg

Females 9 - 18 Years 8 - 15 mg

Children

4 - 8 Years 10 mg

1 - 3 Years 7 mg

7 Months - 1 Year 11 mg

0 - 6 Months 0.27 mg



Dietary Sources of Iron

• Red meat > poultry & fish

• In U.S., 20 mg iron added/lb of flour

• Baked bread contains ~28 mg iron/kg (equivalent to the iron content of beef)

• Iron cooking pots

• Plants are generally not good sources because of oxalate, phytate, tannins, etc.

• Spinach has a lot of iron, but has ~780 mg oxalate/100 g

Note: Heme iron absorption from diet not affected by ascorbate or phytatehttp//ods.od/nih.gov/factssheets/iron 08-24-07



Iron Supplementation in Special Populations

Pregnant Women

• During the last two trimesters, daily iron requirements increase to 5 - 6 mg to maintain normal level

• Normal-term infants are born with sufficient iron stores to prevent iron deficiency for the first 4 - 5 months of life

• Thereafter, enough iron needs to be absorbed to keep pace with the needs of rapid growth

• Nutritional iron deficiency is most common between 6 and 24 months of life

Cochrane Database Syst Rev. Jul 19 2006



What Causes Iron Deficiency?

Increased Iron Needs

• Rapid growth in infancy or adolescence

• Pregnancy

• Erythropoietin therapy

• Acute or chronic Blood loss

• Heavy menstrual periods

• Frequent blood donation

• Some stomach and intestinal conditions (food sensitivity, hookworms)

• Phlebotomy as treatment for Polycythemia Vera

Decreased Iron Intake and Absorption

• Lack of heme iron sources in the diet (e.g., vegetarian diets)

• Low absorption

• Taking antacids beyond the recommended dose or medicine used to treat peptic ulcer disease and acid reflux can reduce the amount of iron absorbed in the stomach

• Malabsorption from disease (Celiac sprue, Crohn's disease)

• Malabsorption from surgery (post-gastrectomy)

• Acute or chronic inflammation

MMWR, April 03, 1998 / 47(RR-3);1-36



Koilonychia(spooning of the

fingernails)

Angular Cheilosis(fissures at the corners

of the mouth)

Glossitis(soreness of the tongue,

or inflammation with depapillation of the dorsal

surface of the tongue)

Esophageal Web(thin membranes

of normal esophageal that can partially obstruct)

Plummer-Vinson Syndrome

Q J Med. Apr 1965;34:145-61

Systemic Manifestation of Iron Deficiency Anemia



Iron Deficiency Anemia

• Hypochromic red cell

• Microcytic cell

• Target cell (Codocytes) in sever cases

• Anisocytosis

• Poikelocytosis

• Mild thrombocytosis

Platt, W.R., Color Atlas and Texbook of Hematology, J.B. Lippincott Company, Philadelphia, PA, 1969:199-201



Chronicrenal failure

Reticulocytehemoglobincontent low

(CHr)

Seen in CRFwho undergo

Tx withepoetin

Serumferritin may

be normal orelevated

One fifth ofpatients starting

dialysis haveabsolute iron

deficiency

Types of Iron Deficiency

Beutler E, et.al. Hematology. 6th ed. New York, NY: McGraw-Hill Book Co; 2001:295-304, 447-70.

Mittman N. et al. Am. J. Kidney Dis. 1997; 30:912-922

AbsoluteIron Deficiency:

Inadequate production of erythropoietin bykidney disease and

decreased totalFe body content

Hemodialysis(Average iron loss57-78 mg/month) Transferrin

saturationusually < 20%,

but variable

FunctionalIron Deficiency:

Iron stores are presentbut cannot be mobilized

rapidly enough tomaintain maximal

erythropoietin-drivenerythropoiesis

ErythrocyteIndicesnormal

%hypochromic

RBC raised( > 5%)



Data suggeststhat high levels

of pro-inflammatorycytokines and increase

oxidative stress arecommon features that

may contribute tomalnutrition, rHuEPO

resistance andanemia

Increasedinternal iron

loss• Gastrointestinal

bleeding

It is largelythe result ofinsufficient

production ofErythropoietin bynon-functioning

kidney

Increasedexternal iron losses

in transfusions:

• Blood loss by the hemodialysis filter & lines

• Frequent phlebotomy for serial monitored iron status

Anemia(i.e. hemoglobin

levels < 11 - 12 g/dL) is almost universalfeature of patients

with end-stagerenal disease

on dialysis

Decreasedavailability of

body’s storageiron

End result– very poor

quality of life

Iron Loss and Iron Deficiency Anemia in ESRD Patients



IronDeficiencyAnemia in

ESRDPatients

Deficitin intestinal

ironabsorption

IronLoss in

End StageRenal

Disease



Measurement of Body Iron Status

Biochemical

• Serum iron (Fe)

• Total iron binding capacity (TIBC)

• Percentage transferrin saturation (%TSAT)

• Soluble circulating transferrin receptor (sTfR)

• Transferrin receptor ratio (TfR/FRT)

• Ferritin

• Haptoglobin

Hematological

• Hgb, MCV,MCH, RDW

• Erythrocyte zinc protoporphyrin (ZPP)

• Reticulocyte Hgb Content (CHr)

• % Hypochromic RBCs (% HYPO)

• Bone marrow iron store





Stages of Iron Deficiency

Tessitore N .et al. Nephr. Dial. Transplant 2001; 16:1416-1423

Depleted Iron Stores Compromised Delivery Iron Deficiency Anemia

Serum ferritin

TSAT

Erythrocyte ZPP

Hb

MCV

% Hypo

CHr



Differential Diagnosis using Iron Panel



MCV Serum Fe TIBC % Saturation Ferritin

Fe Deficiency

ACD N to

Thalassemia

Sidroblastic



Diagnostic Value of Iron Indices in Hemodialysis Patients Receiving Epoetin in Predicting a Response to Iron

James S Kaufman, et al. Kidney International, December 2001, Vol 60, 300-308

Epoetin Dose Change of = 30 U/kg/week Epoetin Dose Change = 60 U/kg/week

True Positives True Negatives Positive

Predictive Value

Negative

Predictive ValueTrue Positives True Negatives

Positive

Predictive Value

Negative

Predictive Value

Serum Ferritin Threshold

<100 ng/mL 13 16 0.76 0.37 10 30 0.63 0.37

<200 ng/mL 29 11 0.76 0.50 19 19 0.53 0.79

<300 ng/mL 37 3 0.68 0.50 22 5 0.42 0.714

<400 ng/mL 40 0 0.67 NA 24 0 0.40 NA

Transferrin Saturation Threshold %

<12 2 17 0.40 0.31 2 33 0.40 0.60

<16 6 13 0.46 0.28 5 28 0.39 0.60

<20 24 6 0.63 0.28 14 12 0.37 0.55

<24 29 4 0.64 0.27 18 9 0.40 0.60

<28 37 4 0.70 0.57 20 8 0.42 0.67



Treatment of Iron Deficiency

• Red blood cell transfusion

• Oral iron therapy

• Ferrous sulfate

• Ferrous fumarate

• Ferrous gluconate

• Parenteral iron

Pediatr Nurs. 2003;29(2).



The Prevalence of Vitamin B-12 Deficiency

Macfarlane et al Am J Clin Nutr 2011;94:1079-1087 ; Pfeiffer et al Am J Clin Nutr 2005;82:442-50 ; Pfeiffer et al Am J Clin Nutr 2007;86:718-27 ; Clarke et al Age Ageing 2004;33:34-41; Stover et al Curr OpinClin Nutr Metab Care 2010;1:24-27

Vitamin B12 deficiency is a global health problem

• In Canada, approximately 5% are B12-deficient.

• In the USA, 3-6% of adults are estimated to have vitamin B12 deficiency (Total B12 <148pmol/L).

• In the UK, B12 deficiency (Total B12 ≤150pmol/L) is estimated at 5% of those aged 65-74y and 10% in those aged ≥75y.

• Marginal depletion (Total B12 of 148-221pmol/L) is more common, occurring in >20% of those aged >60y.

Ability to absorb vitamin B12 decreases with age-associated gastric atrophy therefore prevalence will increase as the global population ages.



Megaloblastic Anemia

Macrocytic RBC

Hypersegmented Neutrophils

• Due to impaired DNA synthesis

• Affects cells primarily having relatively rapid turnover, especially hematopoietic precursors and gastrointestinal epithelial cells

• Cell division is sluggish, but cytoplasmic development progresses normally, so megaloblastic cells tend to be large, with an increased ratio of RNA to DNA.

• Megaloblastic erythroid progenitors tend to be destroyed in the marrow

• Marrow cellularity is often increased but production of red blood cells (RBC) is decreased

Platt,W.R., Color Atlas and Texbook of Hematology, J.B. Lippincott Company, Philadelphia, PA, 1969:203-05

Am Fam Physician. 2003 Mar 1;67(5):979-986.



Pernicious Anemia

• Most common cause of cobalamin deficiency

• Caused by the absence of IF

• Atrophy of the mucosa

• Autoimmune destruction of parietal cells

• Seen in individuals of northern European descent and African Americans

• Men and women are equally affected

• Disease of the elderly, the average patient presenting near age 60

Am J Hematol. 1990;34:99–107



Causes of Megaloblastic/Macrocytic Anemia

Vitamin B12 Deficiency

• Inadequate intake: Vegans (rare)

Malabsorption

• Defective release of cobalamin from food• Gastric achlorhydria-atropic gastritis• Long term drug use that block acid secretion

• Partial gastrectomy• Inadequate production of intrinsic factor (IF)• Pernicious anemia

• Total gastrectomy• Terminal ileal resection (> 100 cm), decreases the

site of absorption of B12-IF complex

• Pancreatic insufficiency

Disorders of Terminal Ileum• Celiac (Sprue) disease• Regional enteritis

• Crohn’s disease

Competition for Cobalamin

• Fish tapeworm (Diphyllobothrium latum)• Bacteria: "blind loop" syndrome• Drugs: p-aminosalicylic acid, colchicine, neomycin

Braunwald E, et.al. Harrison's Principles of Internal Medicine. 15th ed. New York, NY: McGraw Hill; 2001



Causes of Megaloblastic/Macrocytic Anemia

Vitamin B12 Deficiency

• Inadequate intake: Vegans (rare)

Malabsorption

• Defective release of cobalamin from food• Gastric achlorhydria-atropic gastritis• Long term drug use that block acid secretion

• Partial gastrectomy• Inadequate production of intrinsic factor (IF)• Pernicious anemia

• Total gastrectomy• Terminal ileal resection (> 100 cm), decreases the

site of absorption of B12-IF complex

• Pancreatic insufficiency

Disorders of Terminal Ileum• Celiac (Sprue) disease• Regional enteritis

• Crohn’s disease

Competition for Cobalamin

• Fish tapeworm (Diphyllobothrium latum)• Bacteria: "blind loop" syndrome• Drugs: p-aminosalicylic acid, colchicine, neomycin




Clinical Manifestations of Vitamin B12 Deficiency


Hematology (Am Soc Hematol Educ Program). 2003:62-81.

Hematologic

• Macrocytic anemia

• Pancytopenia

Gastrointestinal

• Glossitis

• Anorexia

• Diarrhea

Psychiatric

• Mild irritability and forgetfulness to severe dementia or frank psychosis

Neurologic

(found in 3/4th of individuals with pernicious anemia)

• Paresthesia in the extremities

• Peripheral neuropathy

• Combined systems disease (demyelination of dorsal columns and corticospinal tract)

• Weakness, ataxia, sphincter disturbances



Diagnosis of Vitamin B12 Deficiency

• Macrocytosis

• Peripheral blood smear

• Cobalamin levels

• Serum Vitamin B12

• Elevated serum methylmalonic acid and homocysteine levels

• Serum Folate

• Schilling Test

Am J Hematol. 1990;34:99–107.Am J Med. 1994;96:239–46.



Current Routinely Laboratory Tests

Four biochemical tests routinely used for assessment of Vitamin B12 status:

1. Total Serum B12▪ Measures all circulating B12, both inactive and active forms▪ Significant indeterminate zone where B12 status cannot be confirmed▪ Serious problem with IF-antibody interference

2. Methylmalonic Acid (MMA)▪ B12 deficiency causes elevation▪ Non-specific – also elevated in old age, with renal disease and with gut

bacteria overgrowth

3. Homocysteine▪ B12 deficiency causes elevation▪ Non-specific – also elevated in old age, with renal disease and with folate

deficiency

4. Active B12(Holotranscobalamin or HoloTC)

▪ Transcobalmin transports Vitamin B12 from site of absorption (ileum) to tissues and cells

▪ Vitamin is internalized as Active B12 complex

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3142735/



Schilling Test

Schilling, Robert Frederick (born 1919), American hematologist. Schilling undertook research on the absorption and utilization of vitamin B12, the mechanisms involved in the causation of anemia, and on the gastrointestinal absorption of nutrients. He introduced the Schilling test in 1953.

The Schilling Test• Measures B12 deficiency

• Detects IF deficiency

• Detects abnormal results in patients with genetic defects in B12

absorption, bacterial overgrowth of the small bowel, resection/bypass of terminal ileum, and pancreatic insufficiency

Braunwald E, et.al. Harrison's Principles of Internal Medicine. 15th ed. New York, NY: McGraw Hill; 2001.



Key Points of Vitamin B12 Diagnosis

1. Accurate diagnosis of vitamin B12 status• Diffused and varied signs and symptoms.• Potentially misleading hematological parameters.• Limitations of current front-line Total B12 test and other reflex tests.

2. Early diagnosis of B12 deficiency • Needed in order to initiate treatment before disease symptoms manifest and/or become

irreversible (neurological).

Can Active-B12 address these needs?




Active-B12 vs Vitamin B12

• Active B12 represents 10-30% of Vitamin B12 circulating in the blood.

• Only form of Vitamin B12 taken up and used by the cells of the body.

Vitamin B12 in the Circulation

HolohaptocorrinBiologically Inert70-90%

Active B12(Holotranscobalamin) BiologicallyActive 10-30%

Carmen R and Herbert V, Blood, 1969: 33: 1-12., Hakami N et al., New Eng J Med., 1971; 285: 1163-70, Herzlich B and Herbert V, Lab Invest., 1988; 58: 332-7.



Active-B12 vs Vitamin B12

• The current front-line test, Total B12, measures all of the Vitamin B12 in the blood, but not all circulating B12 can be used by the body.

Specific receptor-mediated

cellular uptake Adenosyl-B12

Met

MMA

Methyl-B12 Hcy

HolotranscobalaminBiologically-activeAround 20% of circulating B12

CobalaminHolohaptocorrinBiologically inertAround 80% of circulating B12

http://www.axis-shield.com/active-b12/



Typical Stages in the Development of a Vitamin B12Deficiency

Clin Chim Acta. 2002 Dec;326(1-2):47-59.

Stage Manifestation Comment

I Circulating serum B12 levels depleted.Patients are typically asymptomatic and

can remain in this stage for several years.

II Cellular stores of B12 are depleted.Patients can remain asymptomatic.

This stage can also continue for several years.

IIIEvidence of bio-chemical deficiency via increases in

serum homocysteine and methylmalonic acid.

Vitamin B12 is required for

the conversion of these compounds.

IV Clinical signs and symptoms apparent.The spectrum of clinical manifestations is broad and

the sequence of symptom development varies markedly.



Laboratory Test Results for the Progression of B-12 Deficiency

Adapted from Herbert V, Nutrition Science as a continually unfolding story: the folate and vitamin B12 paradigm American Journal of Clinical Nutrition 1987; 46: 387-402

Normal B12

status

0 I

Early serumdepletion

II III IV

Celldepletion

Damagedmetabolism

Clinicaldamage

B12 depletionNormal

HoloTCMMA

HomocysteineSerum B12

ErythrocytesMCV

Hb

NormalNormalNormalNormalNormalNormalNormal

LowNormalNormalNormalNormalNormalNormal

LowNormalNormalNormalNormalNormalNormal

LowElevatedElevatedLowNormalNormalNormal

LowElevatedElevatedLowMacrocyticElevatedLow

B12 Deficiency



Correlation of Total B12 with HoloTC

• n=468 patients

• 67% of results are immediately indeterminate by Total B12

• More importantly, 180 patients (38%) have Total B12 levels >150pmol/L but are deficient by HoloTC.

Raw data provided by Prof. W Herrmann, Universitätskliniken des Saarlandes, Homburg, Germany

0

50

100

150

200

250

300

350

0 100 200 300 400 500 600 700 800 900

Ac

tive

-B12

pm

ol/

L

Total B12 pmol/L

n=3

Low

B12,

high

AB12

n=141Grey B12, high AB12

n=95

High B12,

high AB12

n=49

Low B12,

low AB12

n=173

Grey B12,

low AB12

n=7

High B12,

low AB12



New Definition of Vitamin B12 Deficiency

• Use of MMA as the gold standard is flawed due to poor specificity.

• Innovative study in 2011 used Red Blood Cell B12 as a measure of the true B12 status in the tissues and allowed a direct comparison of the power of Active-B12, Total B12 and MMA to detect deficiency.



Performance of Markers vs RBC-B12

• Using this novel approach, Active-B12 has higher AUC and diagnostic performance than Total B12 and MMA

Valente et al Clinical Chemistry 2001 57:6



Performance of Markers in Grey-Zone

• Potential clinical use was assessed by use of a gray-zone with limits of 60% for ruling-in deficiency (PPV) and 98% for ruling-out deficiency (NPV).

MMA HoloTC Total B12

Grey-zone 0.31 to 1.40 µmol/L 19.6 to 29.9 pmol/L 79 to 238 pmol/L

Samples in grey-zone (n)

349/700 96/699 313/700

Samples in grey-zozne (%)

50% 14% 45%

• A high proportion of samples in the gray-zone restricts clinical utility.

Valente et al Clinical Chemistry 2001 57:6



Total B12: Interference from anti-IF Antibodies

• Total B12 test can give False Negatives due to assay interference, as seen in the following articles:

• Failures of Cobalamin assays in Pernicious Anemia Carmel and Agrawal, NEJM 2012 367;4

• Spurious Elevations of Vitamin B12 with Pernicious Anemia Yang and Cook, NEJM 2012 366;18

• Falsely elevated cobalamin concentration in multiple assays in a patient with pernicious anemia: A case study van Rossum et al CCLM 2013 14:1-3



Can Active-B12 Replace Total B12?

If Active-B12 replaces Total B12 as the front line test, the following criteria should be met

• Must be a better marker of Vitamin B12 status.

‒ HoloTC measures only the biologically-available form of B12.

‒ HoloTC appears to be a better measure of B12 status then Total B12 when using RBC-B12 as the true definition.

• Must offer improved sensitivity and specificity.

‒ HoloTC appears to be more sensitive and more specific than Total B12.

• Should have no interference from anti-IF antibodies.

‒ No interference.

• Must be an early marker of deficiency.

‒ Changes in HoloTC levels occur earlier than Total B12 in Vitamin B12 depletion.



Active-B12 in Practice:Resolution of Grey-zone Total B12 Results

• Many users have started using the test as a reflex to Total B12 but as experience with the marker grows, they have stopped Total B12 testing and exclusively use Active-B12.

Adapted from Schneede J., Scan J Clin Lab Invest 2003; 63: 369 – 376

Note that all suggested cut-offs will be dependent on the population served by the laboratory

Subjects at risk of B12 deficiency

Total B12 <150 pmol/L Total B12 150-300 pmol/L Total B12 >300 pmol/L

Measure HoloTC

Likely deficient Unlikely deficient

Resolve B12 indeterminate samples



Active-B12 in Practice:Replacement for Total B12 Assay

• Labs in Australia, Germany, Netherlands and the UK have adopted this approach and no longer use Total B12.

Note that all suggested cut-offs will be dependent on the population served by the laboratory.

Subjects at risk of B12 deficiency

HoloTC <35 pmol/L HoloTC >35 pmol/L

Likely deficient Unlikely deficient**Renal patients should be further investigated with creatinine and/or MMA



Treatment of Vitamin B12 Deficiency

• Replacement therapy

• Parenteral treatment given weekly intramuscularly for 8 weeks, followed by intramuscularly every month for the rest of the patient's life

• Daily oral replacement therapy




Folate Deficiency

Manifestations of Folate Deficiency

More often malnourished than those with

cobalamin deficiency

Gastrointestinal manifestations

More widespread and more severe than those of

pernicious anemia

Diarrhea is often present

Cheilosis (a painful inflammation and cracking of the

corners of the mouth)

Glossitis

Neurologic abnormalities do not occur


Stages of Folate Deficiency

Negative folate balance (decreased serum folate)

Decreased RBC folate levels and

hypersegmented neutrophils

Macroovalocytes, increased MCV, and

decreased hemoglobin



Diagnosis of Folate Deficiency

• Peripheral blood and bone marrow biopsy look exactly like B12

deficiency

• Plasma folate < 3 ng/ml – fluctuates with recent dietary intake

• RBC folate – more reliable of tissue stores < 140 ng/ml

• Only increased serum homocysteinelevels but NOT serum methylmalonic acid levels


http://www.health-writings.com/folate-deficiency-anemia-caused/

Dietary Factors

• Low iron, heme iron

• Excess phytate

• Excess tea or coffee

• Fad diets

Very High

Risk

High Risk

High RiskHigh Risk

Demographic Factors

• Elderly

• Teenager

• Female

Social, Physical Factors

• Poverty

• Poor detention

• Alcohol abuse

• Candle burning

• GIT disease

• Depression

• Immigrant

• Aborigine

• Widower



Treatment of Folate Deficiency

Oral Replacement Therapy

Folate Prophylaxis

• Women planning pregnancy are advised to take 400 g folic acid daily before conception and until 12 weeks of pregnancy to prevent neural-tube defects (5 mg/day for women with a previous affected pregnancy).

• Folate fortification of cereal grains at 1 - 4 mg/kg has been made mandatory in the USA as an additional method of improving the folate status of the population.

• Prophylactic folate is also recommended in other states of increased demand such as long-term hemodialysis and chronic hemolytic disorders.




Inappropriate Treatment of Pernicious AnemiaWith Folate

Vitamin B12 deficiency anemia can be temporarily corrected by folate supplementation

However, this does not correct the neurologic deficits

• Folate “draws” Vitamin B12 away from neurologic system for RBC production and can exacerbate combined systems degeneration




Take Home Message

• Anemia is a widespread public health problem associated with an increased risk of morbidity and mortality, especially in pregnant women and young children.

• Anemia goes undetected in many people, and symptoms can be minor or vague.

• Most commonly, people with anemia report non-specific symptoms of a feeling of weakness, or fatigue, general malaise and sometimes poor concentration.

• Anemia is a disease with multiple causes, both nutritional (Iron, Vitamin B12, Folate deficiencies are most common) and non-nutritional (infection) that frequently co-occur.

• Anemia is a laboratory diagnosis.




Take Home Message

56

• Vitamin B12 deficiency is common in General Practice populations

• Symptoms may be ill-defined and high index of suspicion is necessary

• Vitamin B-12 exists as an active and inactive form in the blood

• All standard B-12 assays measure active and inactive forms and are thus prone to false positives

&negatives. Between 15-40% of patients with low B-12 results do not have B-12 deficiency

• A new "Active B-12” assay measures only active form in measurable in pmole/L and is thus an

improvement for detecting early depletion and deficiency




Thank You!

Questions?



References

1. Kraft, Sy Medical News Today. MediLexicon, Intl., 14 May. 2010. Web: 5 Apr. 2014. http://www.medicalnewstoday.com/articles/188770

2. Int J Gen Med. 2011; 4: 741–750.

3. Veng-Pedersen P, Chapel S, Schmidt RL, Al-Huniti NH, Cook RT, Widness JA. An integrated pharmacodynamic analysis of erythropoietin, reticulocyte, and hemoglobin responses in acute anemia. Pharm Res. Nov 2002;19(11):1630-5.

4. Young NS, Scheinberg P, Calado RT. Aplastic anemia. Curr Opin Hematol. May 2008;15(3):162-8.

5. Adamson JW, Longo DL. Anemia and polycythemia. In: Harrison's Principles of Internal Medicine. Vol 1. 15th ed. New York, New York: McGraw-Hill; 2001:348-354.

6. Mayo Foundation for Medical Education and Research, 1998-2014.

7. Int J Gen Med. 2011; 4: 741–750. http://www.webnat.com/articles/Anemia.asp

8. Hematology. 6th ed. New York, NY: McGraw-Hill Book Co; 2001:295-304, 447-70.

9. Hematology: Basic Principles and Practice. 5th ed. Philadelphia, PA: Churchill Livingstone; 2009:Chap 34:439-46.

10. Int J Gen Med. 2011; 4: 741–750.



References

11. http//ods.od/nih.gov/factssheets/iron 08-24-07.

12. N Engl J Med. 1999;341:1986-1995.

13. Hematology. 6th ed. New York, NY: McGraw-Hill Book Co; 2001:295-304, 447-70.

14. Hoffman R, Benz EJ Jr, Shattil SJ. Hematology: Basic Principles and Practice. Vol 3. New York, NY: Churchill Livingston; 1998:397-427.

15. Mclean E, Cogswell M, Egli I, Wojdyla D, de Benoist B. Public Health Nutrition, May 23: 1-11, 2008.

16. Mason, J. et al. The Micronutrient Database Project. Tulane University, New Orleans, LA, USA Unpublished Data, 2003.

17. McLean E, Public Health Nutrition May 23:1-11, 2008.

19. National Health and Nutrition Examination Survey 2003-2006.

20. Am. J. Obstet. Gynecol., 159: 107-11 ,1988.

21. Cochrane Database Syst Rev. Jul 19 2006.

22. MMWR, April 03, 1998 / 47(RR-3);1-36.



References

23. Q J Med. Apr 1965;34:145-61

24. Platt, W.R., Color Atlas and Texbook of Hematology, J.B. Lippincott Company, Philadelphia, PA, 1969:199-201.

25. Beutler E, et.al. Hematology. 6th ed. New York, NY: McGraw-Hill Book Co; 2001:295-304, 447-70.

26. Mittman N. et al. Am. J. Kidney Dis. 1997; 30:912-922.

27. http://www.cdc.gov/ncbddd/hemochromatosis/training/pathophysiology/iron_cycle_popup.htm

28. Practical Gastroenterology October 2006, 46-64.

29. Tessitore N .et al. Nephr. Dial. Transplant 2001; 16:1416-1423.

30. Am J Kidney Dis. Sep 2009;54(3):498-510.

31. James S Kaufman, et al. Kidney International, December 2001, Vol 60, 300-308.

32. Pediatr Nurs. 2003;29(2).

33. National Health and Nutrition Examination Survey 2001-2004.

34. Am Fam Physician. 2003 Mar 1;67(5):979-986.



References

35. Braunwald E, Fauci AS, Kasper DL, Hauser SL, Longo DL, Jameson JL. Harrison's Principles of Internal Medicine. 15th ed. New York, NY: McGraw Hill; 2001.

36. Hematology (Am Soc Hematol Educ Program). 2003:62-81.

37. Clin Chim Acta. 2002 Dec;326(1-2):47-59.

38. Am J Hematol. 1990;34:99–107.

39. Am J Med. 1994;96:239–46.

40. http://www.health-writings.com/folate-deficiency-anemia-caused/

41. Prchal JT. Production of Erythrocytes. Lichtman MA, Kipps TJ, Seligsohn U, Kaushansky K, Prchal JT, eds. Williams Hematology. 8th ed. McGraw-Hill: New York; 2010. Chapter 31.

42. Means RT, Glader B. General Considerations. Greer JP, Foerster J, Rodgers GM, eds. Anemia: Wintrobe’s Clinical Hematology. 12th ed. Philadelphia, PA: Lippincott Williams and Wilkins; 2009. Vol. 1: Chapter 26.

43. Piva E, Brugnara C, Chiandetti L, Plebani M. Automated reticulocyte counting: state of the art and clinical applications in the evaluation of erythropoiesis. Clin Chem Lab Med. 2010 Oct. 48(10):1369-80. [Medline].

http://www.health-writings.com/folate-deficiency-anemia-caused/

http://reference.medscape.com/medline/abstract/20666695



References

44. Ryan DH. Examination of Blood Cells. Lichtman MA, Kipps TJ, Seligsohn U, Kaushansky K, Prchal JT, eds. Williams Hematology. 2010. http://www.accessmedicine.com/content.aspx?aID=6106433. Accessed. 8th ed. New York: McGraw-Hill; January 15, 2012. Chapter 2.

45. Marks PW, Glader B. Approach to Anemia in the Adult and Child. Hoffman F, Benz EJ, Shattil SJ, eds. Hematology: Basic Principles and Practice. 5th ed. Churchill Livingstone: Philadelpha, PA; 2009. Chapter 34.

46. Torres Gomez A, Casano J, Sanchez J, Madrigal E, Blanco F, Alvarez MA. Utility of reticulocyte maturation parameters in the differential diagnosis of macrocytic anemias. Clin Lab Haematol. 2003 Oct. 25(5):283-8. [Medline].

47. Briggs C, Bain BJ. Basic haematological techniques. Bain BJ, Bates I, Laffan MA, and Lewis SM eds. Dacie and Lewis Practical Haematology. 11th ed. Philadelphia PA: Churchill Livingstone; 2012. chap 3.

48. Brugnara C, Schiller B, Moran J. Reticulocyte hemoglobin equivalent (Ret He) and assessment of iron-deficient states. Clin Lab Haematol. 2006 Oct. 28(5):303-8. [Medline]. [Full Text].

49. Brugnara C, Zurakowski D, DiCanzio J, Boyd T, Platt O. Reticulocyte hemoglobin content to diagnose iron deficiency in children. JAMA. 1999 Jun 16. 281(23):2225-30. [Medline].

50. Karagülle M, Gündüz E, Sahin Mutlu F, Olga Akay M. Clinical significance of reticulocyte hemoglobin content in the diagnosis of iron deficiency anemia. Turk J Haematol. 2013 Jun. 30(2):153-6. [Medline]. [Full Text].



http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618805/



http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3878462/

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