Clin Lab Lecture
For the in class exam you should know:
• Retic Ct.
The in class exam is open note; but not open book— you can bring one thing,
a binder or whatever. You can cram it full of whatever you want— my
handouts, anything. I would allow this to be open book but books take up too
much space and they make a lot of noise during an exam.
Tipson taking this exam successfully:
• Prep as if it were a closed-book exam
• Tab your notes so you can find stuff
• You do not have to memorize normals right now. I’ ll tell you that the
reference values for WBC, for example, are 5000-11,000
• If you are prepped, you’ ll be able to leave early.
There are two parts to your exam:
Part One: The in-class exam on Nov 7
Part Two has two sections, both are due Nov 14
A Flow diagram (please add IDA, it’ s missing from handout)
B Case studies
The key to part A is to be able to utilize it: you must include every one of
these anemias— these are common in a primary care practice.
Please do not pull stuff off the internet or from a book. It’ s okay to start from
a book or website to get your feet wet; but you want to be thinking, “ How
does a PRIMARY CARE doc approach this. NOT a pathologist or lab tech
(who might list bone marrow tap and retic ct. as second test to run).” So
don’ t list every test you can do, list the tests YOU would do.
In addition, you cannot prove a negative. You must have two confirmed tests
for each diagnosis. If you call this “ anemia due to renal disease” , you must
say confirm with renal function tests (you don’ t need to know the names of
tests we haven’ t covered, just “ renal function test” is fine).
If there are two functions of the same anemia, you must follow both. For
example, anemia due to liver disease (dis) can be macro or microcytic, you
must follow both.
I will come up with practice problems for you for next week.
You may not discuss your exam with others. You may talk to me about it.
You can talk about Clin Lab Diag general topics with others; but not exam.
If you want to know that you are heading down the right track, email me and
I will point you in the right direction. Use your best judgment— have
This is excerpted from the patient handout on anemia in MD consult. For me, it helps to have a “big
picture” overview for this lecture.
Megaloblastic anemia is the end product of deficiencies in the B vitamins: folate, and vitamin B12
(also called cobalamin), or both. Such deficiencies produce: abnormally large red blood cells
(megaloblastic) that have a shortened life span. Neurologic problems are also associated with
There are a number of conditions that can cause these deficiencies.
Causes of Vitamin B12 Deficiency:
• Pernicious anemia.
Pernicious anemia is an autoimmune disease in which antibodies are tricked into attacking
stomach’s parietal cells. This results in impaired production of intrinsic factor (IF), a compound
that is critical for absorption of vitamin B12; and HCl.
Pernicious anemia is diagnosed in about 1% of people over 60, with women having a higher
risk than men.
• H. pylori and atrophic gastritis.
A 2000 study suggested that the H. pylori bacterium is a player in many cases of vitamin B12
deficiency. The bacteria are not only major culprits in peptic ulcers, but also are strongly
associated with atrophic gastritis. This condition is a gradual loss of the stomach lining and is a
known cause of vitamin B12 deficiency. (Some researchers theorize that H. pylori-induced
injuries in the stomach lining may actually be the first step in the destructive process that leads
to pernicious anemia.)
• Complications of gastrointestinal surgery.
Surgeries such as stomach bypass or stapling, which remove part or all of the stomach, pose a
15% to 30% chance of causing vitamin B12 deficiencies.
• Overgrowth of intestinal bacteria.
Tropical sprue (an acquired malabsorption disease occurring in tropical climates).
• Overexposure to nitrous oxide.
• Low intake
Vitamin B12 deficiency from diet is very rare, since the liver stores over a three-year supply. It
usually does not occur even in alcoholism, vegetarianism, or in malnourished people with
kidney failure or cancer. Since animal products are the chief source, however, true vegan
vegetarians may need a supplement, fortified food, or appropriate food selection known to
contain adequate amounts of this vitamin.
Causes of Folate Deficiency.
• Intake and Intake +Alcoholism
The body stores only about 100 times its daily requirements for folate and can exhaust this supply
within about three months if the diet is deficient in folate.
Poor diet coupled with alcoholism is the most common cause of folate deficiency. Alcohol abuse
not only contributes to malnutrition, but alcohol causes chemical changes that can result in lower
Any condition that disturbs the small intestine and impairs its absorption ability can cause a
deficiency. Such disorders include the following:
Inflammatory bowel disease
Celiac sprue (a sensitivity reaction to gluten)
Parasitic diseases such as giardiasis
Short bowel syndrome (due to bowel resection)
Deficiencies can also be caused by high demand for folic acid caused by conditions such as:
• Some drugs may also hinder folate absorption
Dilantin for seizures
methotrexate for immunosuppression in Crohn’s and cancer
trimethoprim for bacterial UTI
triamterene for high BP and fluid retention
MEGALOBLASTIC ANEMIAS (MA)
Presentation for this type of anemia is subtle. Unless the pat tells you “I’ve been a vegan
20 y and not taken any vitamin supplements. My chief complaint (CC) is weakness and
fatigue”; you are not going to know off the bat that the pat has MA.
Until you do the labs you may not see a MA pic right away….
The other thing is, folate and B12 deficiency, no matter what they are due to: demand,
intake, pernicious anemia (PA), will look the same.
The only difference in presentation will be that B12 neurological symptoms.
Personality changes, loss of cognition, neuropathy….The anemia is reversible, but the
neurological symptoms may not be.
So if you suspect folate deficiency (def), pls also ck B12 levels.
Folate def can be due to:
• Increased util
• Lo intakenutrient def
B12 def can be due to:
• Lo intake
Clinical Note: In the US and Canada #1 Reason for B12 def is PA
PA is an autoimmune disease (usu in pats with a history of AI dis). Ab formed ag.
stomach parietal cells that make intrinsic factor and HCL or Ab ag. intrinsic factor
(IF) itself. Many people have both. Intrinsic factor is necessary for absorption of B12
in the terminal ileum.
Regardless of cause, B12 def presents as:
Clin symptoms that are evolving over weeks to months
Nutritional def not common you have a big store, takes a while to deplete
PA manifest in middle adulthood 40s and 50s
Parasites suck B12 right out of terminal ileum
Signs and Symptoms include:
2. Shortness of breath
3. Lemon yellow pallor (is usully [usu] mentioned in textbks. but this depends
on skin color)
4. Tongue changes
Inflammation (Infl) of the tongue. B12 is required (req) for maturation of
epithelium (epit) of tongue and rest of GI tract. Tongue will appear raw and
red; or sore, pale and smooth.
6. GI symptoms
usu with PA: abdominal pain, diarrhea, vomiting, nausea.
7. 40-45% will have neurological symptoms at diagnosis.
Fingers tingling and numb, can’t feel where floor is….
Key is getting to Dx of B12 def before neurological symptoms happen. If the cause of vit
B12 def is pernicious anemia, the Ab to the parietal will show up in 75 % and Ab to IF
When we look at hematology changes—and these occur usu before neurological
symptoms you have an opportunity to catch B12 def.
Hematology changes include:
1. Pancytopenia: all the cels are dec in number.
2. WBC : 4-5K, (normal 5-11K/microliter (uL )
3. HCT LO
4. Hb low/normal
Women: mild anemia 10-12
Men: mild anemia 11.5-13
Despite these values, we would still call this macrocytic/normachromic. Why?
Because the cels are bigger, so more Hb can load onto each cel. This gives us
a misleading HI MCH and MCHC. When you actually look at the cels on a
slide they don’t look full of Hb the way a HI MCH normally looks
6. Blood smear:
We would see Ovalocytes oval shaped cels
Macrocytes larger than normal cels
Dacrocytes tear drop shaped cels
This is because, is MA there is an asynchrony bet the
development of the nucleus and the cytoplasm
Anisocytosisvariation in size
Poikilocytosisvariation in shape
Basophilic stipplingaggregate of ribosomes
Howell-Jolly bodiesfragments of DNA
See p.66-86 in text: Practical Hematology for pics of RBC abnormalities and above terms
NP and lymphocytes bigger than usu
Hypersegmented NP: usu 3-4lobesnow 6-10 lobes nuclei
Thrombocytopenia (low platelet ct.) + giant platelets
This is b/c with megakaryocytes, as we remember from last
week polyploidy and thencytoplasm breaks off to form
platelets. But if we have asynchrony as we do with MA,
this development is off and cytoplasm doesn’t pinch off in
time resulting in LO #’s of platelets and plucked off in big
chunks, monster platelets.
B12 def is interfering with DNA. Look at biochemistry
See the following website:
For a tutorial on B12/folate biochemistry
Biochemistry of B12 and its relationship to folate:
Cobalamin is found in the liver of animals bound to protein as methycobalamin or 5'-
deoxyadenosylcobalamin. The vitamin must be hydrolyzed from protein in order to be
Hydrolysis occurs in the stomach by gastric acids; or the intestines by trypsin following
consumption of animal meat. The vitamin is then bound by intrinsic factor, a protein
secreted by parietal cells of the stomach, and carried to the ileum where it is absorbed.
Following absorption the vitamin is transported to the liver in the blood bound to
There are only two clinically significant reactions in the body that require vitamin B12 as
a cofactor. During the catabolism of fatty acids, the amino acids valine, isoleucine and
threonine and the resultant propionyl-CoA is converted to succinyl-CoA for oxidation in
the TCA cycle. One of the enzymes in this pathway, methylmalonyl-CoA mutase,
requires vitamin B12 as a cofactor in the conversion of methylmalonyl-CoA to succinyl-
The second reaction requiring vitamin B12 catalyzes the conversion of homocysteine to
methionine and is catalyzed by methionine synthase. This reaction results in the transfer
of the methyl group from N5-methyltetrahydrofolate to hydroxycobalamin generating
tetrahydrofolate (THF) and methylcobalamin during the process of the conversion.
Several forms of folate must pass through THF. The form trapped by a B12 deficiency is
N5-methyl THF. Without B12, this form of folate cannot donate it’s methyl group to
make the necessary folate precursor. So with a B12 def, a significant portion of the
body’s folate winds up in a dead end form and becomes useless.
Gastric pH Test
With vit B12 def, the epit in the GI tract is not developing normally. In addition, if this
def is due to PA there is the inhibition of HCl: hypochloridic. To test for low levels of
HCl, you would do a gastric pH test.
In this test, you swallow a capsule with a string attached, it goes into the stomach, the
color changes depending on the pH. After a certain time, you pull it up and read the color.
The Heidelberg capsule is a version of this test that uses a capsule with a radio transmitter
on ittransmit pH. The Heidelberg capsule is little more specific.
Another way to test gastric pH is to administer sodium bicarbarbonate or food prior to
giving capsule and see reaction.
Low moderate sensitivity with both tests.
Scope has a higher sensitivity but is better for lesions
Clinical notes:Some NDs will skip the whole thing and administer HCl and enzymes
Stool sample: O& P Test
B12 def could be due to intake, PA, malabsorption and parasites:
Let’s talk about parasites. Parasites are common in developing countries and Scandavian
countries ( b/c raw fish consumption)
So what is common in these cases is a fish tapeworm. Worldwide, this is the leading
cause of vitamin B12 def : diphyllobothrium latum
If person has just returned from overseas, you can do an O & P Test (Ova and Parasites):
and you should be doing this with three consecutive stool samples on a symptomatic
1st Stool sample60% sens
3 stool samples consecutive on a symptomatic pat 80-85% sens
If per is asymptomatic 40% sens
Tests/Labs/Hx to Isolate etiology of MA as malabsorption:
Three conditions to look out for:
8. Celiac Sprue
9. Long term effective gastric bypass
In these cases, you are not going to see only Vitamin B12 def. Vit D, E, A, K may also be
def. The result would be reduced night vision, increased bruising and ptikiai (sp?). There
may also be poor fat absorption resulting in stool that floats or is malodorous. There may
be blood in the stool resulting in black, tarry, sticky stool. There may also be bloody
diarrhea and abdominal cramping/ pain
CBC won’t just be macrocytic or microcytic due to iron def, there will be coagulation
Dx of IBD:
Scope: sigmoid, colon
If intake: do HX
Following the Yellow Brick Road, the labs to differentiate
between vit B12/folate def and their etiology
If based on the CBC and Hx/presentation, you suspect folate and B12 def, you may
choose to do the following:
1. Serum folate
a. low sensitivity
b. not a good test
i. snapshot is dependent on what you just consumed
2. RBC folate
a. moderately sens b/c RBC live for 120 days.
Clinical note: I would not recommend serum. I would recommend
RBC and VERY GOOD Hx including very good diet history.
Vit B12 def
1. Serum B12
2. Methyl Malonic (MM) acid measured in urine
a. methyl malonyl CoA succinyl CoA in the presence of Vit B12. If no
B12 go directly to MM acid. Higher amts of MM acid are correlated to def
3. Homocysteine levels measured in the serum
a. HS Methionine in the presence of B12 and folate. So if you have a def
in either, you’ll end up with higher amts of HS. This is a serum test. So
serum HS levels will be higher with decreasing levels of B12 and folate.
Clinical Note: Not one good test for vitB12 def so run 2/3
But we still haven’t discussed a way to differentiate the cause of B12: demand, intake
etc. For this, we have the highly moderate sensitivity test, the Schilling test, which will
tell you the etiology of your def.
We begin with a suspicion that pat is B12 def, even if we are running low/normal levels
of other values. We give radioactive B12 (colbalt) in excess orally. Assuming normal
renal function, this is absorbed circulationexcreted in kidney. We are looking for
radioactive B12 in the urine.
Result of Schilling Etiology
If we excrete appropriate amt LO intake of B12/folate
If we don’t excrete it Malabsorption and/or PA
To differentiate between malabsorption and PA, take radioactive B12 + IF in excess
orally. Again measure urine output.
Result of Schilling Etiology
If we excrete normal or greater amts LO IF PA
If we excrete low levels Malabsorption
Why the 1st result? Because you dump in so much IF, you have outnumbered the
antibodies available to block IF. A parallel is with diabetics. Over the years, b/c of insulin
intake you develop antibodies to insulin and have to take larger doses to overcome the
inhibitory effect of those antibodies.
Your Schilling results should be reconfirmed. Parasites will not be not detected with
Recap of Megaloblastic Anemia
Pat walks in with CC of weakness and fatigue.
Serum Vit B12
Or MM acid in urine
Or HS levels
Other things to look at might be:
Tests for malabsorption like fatty stool, intestinal biopsy, scope, vitamin A, or iron, or
I don’t expect you to use all these tests—think logically.
How do you treat PA?
Naturopathic: HCl + Digestive enzymes; regular B12 injections and nutritional stuff to
Would it help to administer long term IF?
I don’t know, the vit B12 injection may aggravate AI dis so it’s something worth
Take Home Message
MA implies an asynchrony in development between the cytoplasm and nucleus.
B12 is needed for all developing cels in body: hematopoietic and GI epit. I don’t
remember mechanism of neurological damage, it has something to do with FA synthesis.
Anemias associated with interference in Synthesis
Iron Deficiency Anemia and Anemia of Chronic Disease
Number One Anemia is ACD usu found in in-patient clinics
Number two Anemia is IDA, usu found in out-patient
Number One nutrition def is IDA
Iron depleted, no longer sufficient store.
Total amt of Iron in body 4g
In circ 60%
Two forms it’s stored in Ferritin and hemosiderin
Where is it stored Liver and Bone marrow (bm)
What stores it Macrophages
Amt of blood we lose a day 25 ml
Amt of blood recycled/day 24 ml
Net loss of iron/day 1 mg
Avg intake of daily iron in our diets 3-5 mg
So if you were a man, short of a blood loss like a GI bleed or cancer, you wouldn’t really
lose iron. Good multis for men don’t have iron because it leads to iron loading other
Women lose iron every month and in pregnancy will have increased util.
Presentation with IDA
So things you want to think about when you do your history:
• is this a child
• is this a pregnant woman
• Nutrit def and loss
• menstrual in women
• Other losses in women and men.
Again for malabsorption, you want to ask questions about
• night vision
• easy bruising
• ptikyi (sp?)
• quality of stool
Clinical note: When you ask, “Are your periods heavy?” Most women will say ‘yes’. You
need to ask, “how long is your period, and how often do you go through tampons etc”
Ask about the GI tract:
• Blood in stool origin is lower down
• Tarry, sticky, black stool origin is high up possible stomach/duod ulcers
• Ulcers prior Hx
• Lots of aspirin ?
• Drug history
Labs for IDA
Iron def is viewed in three stages:
Proactive heading that way, showing predisposition to signs and symptoms
1st stage: iron stores decreased.
Ferritin serum normals have become wider and wider.
Low/normal iron stores
No real signs and symptoms at this point and hematology symptoms are normal. But as a
primary care doc you are trying to prevent stuff sot hink about how to get more dietary
We have progressed used our iron in circ and started deep into the stores.
Some signs and symptoms will include:
• Post running, hard to catch breath
• Tires easily
Pretty normal; but slight changes
HCT 38- 39
Serum IRON Normal slightly Low (when you are
down, stores dump so this won’t read very
Hb Slightly LO
Indices Might start to have a dimorphic pop. RBCs
live 120 days, and less iron to play with
Microcytic/NChrom to NCytic/NChrom
Serum ferritin LO
Your HCT usu runs about 42, but now 38, 39 trending down. Slight, slight abnormal;
but if you don’t know individual reference values.. you may not catch this.
Classic iron def. Anemia considered moderate to severe
Clinical Guideline for Hb:
slight anemia 10-12
severe: below 8
Severe : below 9
Serum iron LO
Serum ferritin LO
Note: Can differentiate bet IDA and ACD with lab tests for:
Transferrin (TF) is a transport protien made by liver. TF levels increase in iron def. TF is
a bus, intended to take iron from one place to another. Normally this bus is half full. If we
have more buses made and less iron riding the buses, TBIC total iron binding capacity
will be increased. I think this the best test, b/c it’s the most sens.
Transferrin will usu be the opposite of TBIC because it is calculated as:
Serum Fe x 100/ TIBC. And transferring will be low in IDA.
Note: You can use all three readings or just one. And by the way, liver dis will f$ck
up the picture.
Anemia of Chronic Dis
IDA and ACD mimic each other.
Big picture in ACD:
Acute and chronic inflammationIL-1, IL-6 and TNFsequestration of Fe and
increased transport from circ back to stores.
In an acute infection, bacteria use Fe, so there may be an evolutionary benefit to this
Initial pic of ACD looks IDA. The big difference is in the iron stores.