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Overview of Anemia:Diagnostic Testingin the Clinical &Laboratory MedicineMonet N. Sayegh, M.D.Physician Consultant
Monet N. Sayegh, M.D
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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.
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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
Monet N. Sayegh, M.D
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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%
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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
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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
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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
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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
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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
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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
Adamson JW, et.al. Harrison's Principles of Internal Medicine. Vol 1. 15th ed. New York, New York: McGraw-Hill; 2001:348-354
Unaffected“Carrier”
Father
Unaffected“Carrier”Mother
Unaffected1 in 4 chance
Unaffected “Carrier”2 in 4 chance
Affected1 in 4 chance
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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
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Pulmonaryinsufficiency
Tissuehypoxia Shock
Complications of Severe Anemia
Adamson JW, et.al. Harrison's Principles of Internal Medicine. Vol 1. 15th ed. New York, New York: McGraw-Hill; 2001:348-354
Themost serious
complications ofsevere anemia
arise from
Hypotension
Coronaryinsufficiency
Death
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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
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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
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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
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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
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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
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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
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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
Monet N. Sayegh, M.D
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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
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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%)
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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
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
IronDeficiencyAnemia in
ESRDPatients
Deficitin intestinal
ironabsorption
IronLoss in
End StageRenal
Disease
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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
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
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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
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Differential Diagnosis using Iron Panel
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
MCV Serum Fe TIBC % Saturation Ferritin
Fe Deficiency
ACD N to
Thalassemia
Sidroblastic
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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
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Treatment of Iron Deficiency
• Red blood cell transfusion
• Oral iron therapy
• Ferrous sulfate
• Ferrous fumarate
• Ferrous gluconate
• Parenteral iron
Pediatr Nurs. 2003;29(2).
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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.
Monet N. Sayegh, M.D
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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.
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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
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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
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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
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Clinical Manifestations of Vitamin B12 Deficiency
Braunwald E, et.al. Harrison's Principles of Internal Medicine. 15th ed. New York, NY: McGraw Hill; 2001
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
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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.
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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/
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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.
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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?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3142735/
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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.
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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/
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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.
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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
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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
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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.
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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
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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
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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
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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.
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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
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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
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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
Braunwald E, et.al. Harrison's Principles of Internal Medicine. 15th ed. New York, NY: McGraw Hill; 2001.
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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
Braunwald E, et.al. Harrison's Principles of Internal Medicine. 15th ed. New York, NY: McGraw Hill; 2001.
Stages of Folate Deficiency
Negative folate balance (decreased serum folate)
Decreased RBC folate levels and
hypersegmented neutrophils
Macroovalocytes, increased MCV, and
decreased hemoglobin
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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
Braunwald E, et.al. Harrison's Principles of Internal Medicine. 15th ed. New York, NY: McGraw Hill; 2001.
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
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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.
Braunwald E, et.al. Harrison's Principles of Internal Medicine. 15th ed. New York, NY: McGraw Hill; 2001.
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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
Braunwald E, et.al. Harrison's Principles of Internal Medicine. 15th ed. New York, NY: McGraw Hill; 2001.
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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.
Braunwald E, et.al. Harrison's Principles of Internal Medicine. 15th ed. New York, NY: McGraw Hill; 2001.
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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
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3142735/
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Thank You!
Questions?
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References
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