The gold standard for the diagnosis of Vitamin B deficiency is a low blood level of Vitamin B. A low level of blood Vitamin B is a finding that normally can and should be treated by injections, supplementation, or dietary or lifestyle advice, but it is not a diagnosis. Hypovitaminosis B can result from a number of mechanisms, including those listed above. For determination of cause, further patient history, testing, and empirical therapy may be clinically indicated.

A measurement of methylmalonic acid (methylmalonate) can provide an indirect method for partially differentiating Vitamin B and folate deficiencies. The level of methylmalonic acid is not elevated in folic acid deficiency. Direct measurement of blood cobalamin remains the gold standard because the test for elevated methylmalonic acid is not specific enough. Vitamin B is one necessary prosthetic group to the enzyme methylmalonyl-coenzyme A mutase. Vitamin B deficiency is but one among the conditions that can lead to dysfunction of this enzyme and a buildup of its substrate, methylmalonic acid, the elevated level of which can be detected in the urine and blood.

Due to the lack of available radioactive Vitamin B, the Schilling test is now largely a historical artifact. The Schilling test was performed in the past to help determine the nature of the vitamin B deficiency. An advantage of the Schilling test was that it often included Vitamin B with intrinsic factor.

The National Institutes of Health has found that "Large amounts of folic acid can mask the damaging effects of vitamin B deficiency by correcting the megaloblastic anemia caused by vitamin B deficiency without correcting the neurological damage that also occurs", there are also indications that "high serum folate levels might not only mask vitamin B deficiency, but could also exacerbate the anemia and worsen the cognitive symptoms associated with vitamin B deficiency". Due to the fact that in the United States legislation has required enriched flour to contain folic acid to reduce cases of fetal neural-tube defects, consumers may be ingesting more than they realize. To counter the masking effect of B deficiency the NIH recommends "folic acid intake from fortified food and supplements should not exceed 1,000 μg daily in healthy adults." Most importantly, B deficiency needs to be treated with B repletion. Limiting folic acid will not counter the irrevocable neurological damage that is caused by untreated B deficiency.

Serum B levels are often low in B deficiency, but if other features of B deficiency are present with normal B then further investigation is warranted. One possible explanation for normal B levels in B deficiency is antibody interference in people with high titres of intrinsic factor antibody.

Some researchers propose that the current standard norms of vitamin B levels are too low.

One Japanese study states the normal limits as 500–1,300 pg/mL. Range of vitamin B12 levels in humans is considered as normal: >300 pg/mL; moderate deficiency: 201–300 pg/mL; and severe deficiency: <201 pg/mL.

Serum vitamin B tests results are in pg/mL (picograms/milliliter) or pmol/L (picomoles/liter). The laboratory reference ranges for these units are similar, since the molecular weight of B is approximately 1000, the difference between mL and L. Thus: 550 pg/mL = 400 pmol/L.

Serum homocysteine and methylmalonic acid levels are considered more reliable indicators of B deficiency than the concentration of B in blood. The levels of these substances are high in B deficiency and can be helpful if the diagnosis is unclear.

Routine monitoring of methylmalonic acid levels in urine is an option for people who may not be getting enough dietary B, as a rise in methylmalonic acid levels may be an early indication of deficiency.

If nervous system damage is suspected, B analysis in cerebrospinal fluid is possible, though such an invasive test should be considered only if blood testing is inconclusive.

The Schilling test has been largely supplanted by tests for antiparietal cell and intrinsic factor antibodies.

PA may be suspected when a patient's blood smear shows large, fragile, immature erythrocytes, known as megaloblasts. A diagnosis of PA first requires demonstration of megaloblastic anemia by conducting a full blood count and blood smear, which evaluates the mean corpuscular volume (MCV), as well the mean corpuscular hemoglobin concentration (MCHC). PA is identified with a high MCV (macrocytic anemia) and a normal MCHC (normochromic anemia). Ovalocytes are also typically seen on the blood smear, and a pathognomonic feature of megaloblastic anemias (which include PA and others) is hypersegmented neutrophils.

Serum vitamin B levels are used to detect its deficiency, but they do not distinguish its causes. Vitamin B levels can be falsely high or low and data for sensitivity and specificity vary widely. Normal serum levels may be found in cases of deficiency where myeloproliferative disorders, liver disease, transcobalamin II deficiency, or intestinal bacterial overgrowth are present. Low levels of serum vitamin B may be caused by other factors than B deficiency, such as folate deficiency, pregnancy, oral contraceptive use, haptocorrin deficiency, and myeloma.

The presence of antibodies to gastric parietal cells and intrinsic factor is common in PA. Parietal cell antibodies are found in other autoimmune disorders and also in up to 10% of healthy individuals, making the test nonspecific. However, around 85% of PA patients have parietal cell antibodies, which means they are a sensitive marker for the disease. Intrinsic factor antibodies are much less sensitive than parietal cell antibodies, but they are much more specific. They are found in about half of PA patients and are very rarely found in other disorders. These antibody tests can distinguish between PA and food-B malabsorption. The combination of both tests of intrinsic factor antibodies and parietal cell antibodies may improve overall sensitivity and specificity of the diagnostic results.

A buildup of certain metabolites occurs in B deficiency due to its role in cellular physiology. Methylmalonic acid (MMA) can be measured in both the blood and urine, whereas homocysteine is only measured in the blood. An increase in both MMA and homocysteine can distinguish between B deficiency and folate deficiency because only homocysteine increases in the latter.

Elevated gastrin levels can be found in around 80-90% of PA cases, but they may also be found in other forms of gastritis. Decreased pepsinogen I levels or a decreased pepsinogen I to pepsinogen II ratio may also be found, although these findings are less specific to PA and can be found in food-B malabsorption and other forms of gastritis.

The diagnosis of atrophic gastritis type A should be confirmed by gastroscopy and stepwise biopsy. About 90% of individuals with PA have antibodies for parietal cells; however, only 50% of all individuals in the general population with these antibodies have pernicious anemia.

Forms of vitamin B deficiency other than PA must be considered in the differential diagnosis of megaloblastic anemia. For example, a B-deficient state which causes megaloblastic anemia and which may be mistaken for classical PA may be caused by infection with the tapeworm "Diphyllobothrium latum", possibly due to the parasite's competition with host for vitamin B.

The classic test for PA, the Schilling test, is no longer widely used, as more efficient methods are available. This historic test consisted, in its first step, of taking an oral dose of radiolabelled vitamin B, followed by quantitation of the vitamin in the patient's urine over a 24-hour period via measurement of the radioactivity. A second step of the test repeats the regimen and procedure of the first step, with the addition of oral intrinsic factor. A patient with PA presents lower than normal amounts of intrinsic factor; hence, addition of intrinsic factor in the second step results in an increase in vitamin B absorption (over the baseline established in the first). The Schilling test distinguished PA from other forms of B deficiency, specifically, from Imerslund-Grasbeck Syndrome (IGS), a vitamin B12-deficiency caused by mutations in the cobalamin receptor.

The blood film can point towards vitamin deficiency:

- Decreased red blood cell (RBC) count and hemoglobin levels

- Increased mean corpuscular volume (MCV, >100 fL) and mean corpuscular hemoglobin (MCH)

- Normal mean corpuscular hemoglobin concentration (MCHC, 32–36 g/dL)

- The reticulocyte count is decreased due to destruction of fragile and abnormal megaloblastic erythroid precursor.

- The platelet count may be reduced.

- Neutrophil granulocytes may show multisegmented nuclei ("senile neutrophil"). This is thought to be due to decreased production and a compensatory prolonged lifespan for circulating neutrophils, which increase numbers of nuclear segments with age.

- Anisocytosis (increased variation in RBC size) and poikilocytosis (abnormally shaped RBCs).

- Macrocytes (larger than normal RBCs) are present.

- Ovalocytes (oval-shaped RBCs) are present.

- Howell-Jolly bodies (chromosomal remnant) also present.

Blood chemistries will also show:

- An increased lactic acid dehydrogenase (LDH) level. The isozyme is LDH-2 which is typical of the serum and hematopoetic cells.

- Increased homocysteine and methylmalonic acid in Vitamin B deficiency

- Increased homocysteine in folate deficiency

Normal levels of both methylmalonic acid and total homocysteine rule out clinically significant cobalamin deficiency with virtual certainty.

Bone marrow (not normally checked in a patient suspected of megaloblastic anemia) shows megaloblastic hyperplasia.

Since the essential pathology is due to the inability to absorb vitamin B from the bowels, the solution is therefore injection of IV vitamin B. Timing is essential, as some of the side effects of vitamin B deficiency are reversible (such as RBC indices, peripheral RBC smear findings such as hypersegmented neutrophils, or even high levels of methylmalonyl CoA), but some side effects are irreversible as they are of a neurological source (such as tabes dorsalis, and peripheral neuropathy). High suspicion should be exercised when a neonate, or a pediatric patient presents with anemia, proteinuria, sufficient vitamin B dietary intake, and no signs of pernicious anemia.

The treatment of PA varies by country and area. Opinions vary over the efficacy of administration (parenteral/oral), the amount and time interval of the doses, or the forms of vitamin B (e.g. cyanocobalamin/hydroxocobalamin). More comprehensive studies are still needed in order to validate the feasibility of a particular therapeutic method for PA in clinical practices. A permanent cure for PA is lacking, although repletion of B should be expected to result in cessation of anemia-related symptoms, a halt in neurological deterioration, and in cases where neurological problems are not advanced, neurological recovery and a complete and permanent remission of all symptoms, so long as B is supplemented. Repletion of B can be accomplished in a variety of ways.

The prevalence of vitamin K deficiency varies by geographic region. For infants in the United States, vitamin K deficiency without bleeding may occur in as many as 50% of infants younger than 5 days old, with the classic hemorrhagic disease occurring in 0.25-1.7% of infants. Therefore, the Committee on Nutrition of the American Academy of Pediatrics recommends that 0.5 to 1.0 mg Vitamin K be administered to all newborns shortly after birth.

Postmenopausal and elderly women in Thailand have high risk of Vitamin K deficiency, compared with the normal value of young, reproductive females.

Current dosage recommendations for Vitamin K may be too low. The deposition of calcium in soft tissues, including arterial walls, is quite common, especially in those suffering from atherosclerosis, suggesting that Vitamin K deficiency is more common than previously thought.

Because colonic bacteria synthesize a significant portion of the Vitamin K required for human needs, individuals with disruptions to or insufficient amounts of these bacteria can be at risk for Vitamin K deficiency. Newborns, as mentioned above, fit into this category, as their colons are frequently not adequately colonized in the first five to seven days of life. (Consumption of the mother's milk can undo this temporary problem.) Another at-risk population comprises those individuals on any sort of long-term antibiotic therapy, as this can diminish the population of normal gut flora.

This is a rare disease with prevalence about 1 in 200,000 to 1 in 600,000. Studies showed that mutations in "CUBN" or "AMN" clustered particularly in the Scandinavian countries and the Eastern Mediterranean regions. Founder effect, higher clinical awareness to IGS, and

frequent consanguineous marriages all play a role in the higher prevalence of IGS among these populations

Diagnosis of inherited hypoprothrombinemia, relies heavily on a patient's medical history, family history of bleeding issues, and lab exams performed by a hematologist. A physical examination by a general physician should also be performed in order to determine whether the condition is congenital or acquired, as well as ruling out other possible conditions with similar symptoms. For acquired forms, information must be taken regarding current diseases and medications taken by the patient, if applicable.

Lab tests that are performed to determine diagnosis:

1. Factor Assays: To observe the performance of specific factors (II) to identify missing/poorly performing factors. These lab tests are typically performed first in order to determine the status of the factor.

2. Prothrombin Blood Test: Determines if patient has deficient or low levels of Factor II.

3. Vitamin K1 Test: Performed to evaluate bleeding of unknown causes, nosebleeds, and identified bruising. To accomplish this, a band is wrapped around the patient's arm, 4 inches above the superficial vein site in the elbow pit. The vein is penetrated with the needle and amount of blood required for testing is obtained. Decreased vitamin K levels are suggestive of hypoprothrombinemia. However, this exam is rarely used as a Prothrombin Blood Test is performed beforehand.

Blood tests are neede to differentiate FVII deficiency from other bleeding disorders. Typical is a discordance between the prolonged prothrombin time (PT) and normal levels for the activated partial thromboplastin time (APTT). FVII levels are <10IU/dl in homozygous individuals, and between 20-60 in heterozygous carriers. The FCVII: C assay supports the diagnosis.

The FVII gene (F7) is found on chromosome 13q34. Heterogeneous mutations have been described in FVII deficient patients.

In congenital FXII deficiency treatment is not necessary. In acquired FXII deficiency the underlying problem needs to be addressed.

The condition is diagnosed by blood tests in the laboratory when it is noted that special blood clotting test are abnormal. Specifically prothrombin time (PT) or activated partial thromboplastin time(aPTT) are prolonged. The diagnosis is confirmed by an assay detecting very low or absent FXII levels.

The FXII (F12) gene is found on chromosome 5q33-qter.

In hereditary angioedema type III an increased activity of factor XII has been described.

Menaquinone (vitamin K), but not phylloquinone (vitamin K), intake is associated with reduced risk of CHD mortality, all-cause mortality and severe aortic calcification.

The differential diagnosis for this inherited condition is the following: hemophilia A, factor XI deficiency, von Willebrand disease, fibrinogen disorders and Bernard-Soulier syndrome

A 28 month old girl, showed symptoms from 8 months of age and consisted of complaints of painful bruises over lower limbs, and disturbed, painful sleep at night. Family history revealed older brother also suffered similar problems and died at age of two years possibly due to bleeding - no diagnosis was confirmed. Complete blood count and blood smear was determined as normal. No abnormality in fibrinogen, liver function test, and bleeding time. However, prothrombin levels were less than 1% so patient was transfused with fresh frozen plasma (FFP). Post transfusion methods, patient is now 28 months old and living healthy life. The only treatment that is needed to date is for the painful bruises, which the patient is given FFP every 5-6 weeks.

Twelve day old boy admitted for symptoms consisting of blood stained vomiting and dark colored stool. Upon admission into hospital, patient received vitamin K and FFP transfusion. No family history of similarity in symptoms that were presented. At 40 days old, patient showed symptoms of tonic posturing and constant vomiting. CT scan revealed subdural hemorrhage, and other testing showed low hb levels of 7%, platelets at 3.5 lakhs/cu mm. PT examination was 51 seconds and aPTT at 87 seconds. Prothrombin activity levels were less than 1%. All other exams revealed no abnormalities. Treatment methods included vitamin K and FFP, as well as ventilator support and packed red blood cell transfusion (PRBC). At half a year of age, condition consisted of possible poor neurological outcome secondary to CNS bleeding. Treatment of very frequent transfusion was needed for patient.

Recent study illustrated a patient with 2 weeks of continuous bleeding, with presence of epistaxis, melena, hematuria, and pruritic rash with no previous bleeding history. Vitals were all within normal range, however, presence of ecchymoses was visible in chest, back and upper areas. Lab exams revealed prolonged prothrombin time (PT) of 34.4 and acquired partial thromboplastin time (aPTT) of 81.7, as well as elevated liver function tests. Discontinuation of atorvastatin, caused liver enzymes to go back to normal. Treatment of vitamin K, antibiotics, and fresh frozen plasma (FFP) did not have an impact on coagulopathy. Mixing of PT and aPTT was performed in order to further evaluate coagulopathy and revealed no correction. Factor activity assays were performed to determine the presence of a specific one. Testing revealed that factor II activity could not be quantified. Further studies showed that acquired factor II inhibitor was present without the lupus anticoagulant, with no clear cause associated with the condition. Aimed to control bleeding and getting rid of the inhibitor through directly treating the underlying disease or through immunosuppressive therapy. Corticosteroids and intravenous immunoglobulin improved the PT and aPTT. Did not improve bleeding conditions until treatment of transfusion with activated PCC. Treatment of inhibitor required Rituximab, which was shown to increase factor II levels to 264%. Study shows that when a patient with no history of coagulopathy presents themselves with hemorrhagic diathesis, direct testing of a factor II inhibitor should be performed initially.

There are several treatments available for factor VII deficiency; they all replace deficient FVII.

1. Recombinant FVIIa concentrate (rFVIIa) is a recombinant treatment that is highly effective and has no risk of fluid overload or viral disease. It may be the optimal therapy.

2. Plasma derived Factor VII concentrate (pdFVII) : This treatment is suitable for surgery but can lead to thrombosis. It is virus attenuated.

3. Prothrombin complex concentrate (PCC) containing factor VII: this treatment is suitable for surgery, but has a risk of thrombosis. It is virus attenuated.

4. Fresh frozen plasma (FFP): This is relatively inexpensive and readily available. While effective this treatment carries a risk of blood-borne viruses and fluid overload.

Two of the most common differential diagnoses are haemophilia B which is a deficiency in Factor IX and von Willebrand Disease which is a deficiency in von Willebrand factor (needed for the proper functioning of Factor VIII), haemophilia C is also a possible, differential diagnosis.

The diagnosis for hemophilia B can be done via the following tests/methods:

- Coagulation screening test

- Bleeding scores

- Coagulation factor assays

Photomutilation and transfusion dependent anemia are common complications. Liver disease is also observed in some cases. It has been reported that early childhood-onset haematological manifestations is a poor prognosis factor.

The diagnosis of hemophilia A may be suspected as coagulation testing reveals an increased PTT in the context of a normal PT and bleeding time. PTT tests are the first blood test done when haemophilia is indicated. However, the diagnosis is made in the presence of very low levels of Factor VIII. A family history is frequently present, although not essential. Recently, genetic testing has been made available to determine an individual's risk of attaining or passing on hemophilia. Diagnosis of haemophilia A also includes a severity level which can range from mild to severe based on the amount of active and functioning factor VIII detected in the blood. Factor VIII levels do not typically change throughout an individual's life. Severe haemophilia A is the most common form occurring in the majority of affected people. Individuals with mild haemophilia often experience few or no bleeding episodes except in the case of serious trauma (i.e. tooth extraction, or surgery).

The diagnosis of hemolytic anemia can be suspected on the basis of a constellation of symptoms and is largely based on the presence of anemia, an increased proportion of immature red cells (reticulocytes) and a decrease in the level of haptoglobin, a protein that binds free hemoglobin. Examination of a peripheral blood smear and some other laboratory studies can contribute to the diagnosis. Symptoms of hemolytic anemia include those that can occur in all anemias as well as the specific consequences of hemolysis. All anemias can cause fatigue, shortness of breath, decreased ability to exercise when severe. Symptoms specifically related to hemolysis include jaundice and dark colored urine due to the presence of hemoglobin (hemaglobinuria). When restricted to the morning hemaglobinuria may suggest paroxysmal nocturnal haemoglobinuria. Direct examination of blood under a microscope in a peripheral blood smear may demonstrate red blood cell fragments called schistocytes, red blood cells that look like spheres (spherocytes), and/or red blood cells missing small pieces (bite cells). An increased number of newly made red blood cells (reticulocytes) may also be a sign of bone marrow compensation for anemia. Laboratory studies commonly used to investigate hemolytic anemia include blood tests for breakdown products of red blood cells, bilirubin and lactate dehydrogenase, a test for the free hemoglobin binding protein haptoglobin, and the direct Coombs test to evaluate antibody binding to red blood cells suggesting autoimmune hemolytic anemia.

The treatment of primary immunodeficiencies depends foremost on the nature of the abnormality. Somatic treatment of primarily genetic defects is in its infancy. Most treatment is therefore passive and palliative, and falls into two modalities: managing infections and boosting the immune system.

Reduction of exposure to pathogens may be recommended, and in many situations prophylactic antibiotics or antivirals may be advised.

In the case of humoral immune deficiency, immunoglobulin replacement therapy in the form of intravenous immunoglobulin (IVIG) or subcutaneous immunoglobulin (SCIG) may be available.

In cases of autoimmune disorders, immunosuppression therapies like corticosteroids may be prescribed.

The basic tests performed when an immunodeficiency is suspected should include a full blood count (including accurate lymphocyte and granulocyte counts) and immunoglobulin levels (the three most important types of antibodies: IgG, IgA and IgM).

Other tests are performed depending on the suspected disorder:

- Quantification of the different types of mononuclear cells in the blood (i.e. lymphocytes and monocytes): different groups of T lymphocytes (dependent on their cell surface markers, e.g. CD4+, CD8+, CD3+, TCRαβ and TCRγδ), groups of B lymphocytes (CD19, CD20, CD21 and Immunoglobulin), natural killer cells and monocytes (CD15+), as well as activation markers (HLA-DR, CD25, CD80 (B cells).

- Tests for T cell function: skin tests for delayed-type hypersensitivity, cell responses to mitogens and allogeneic cells, cytokine production by cells

- Tests for B cell function: antibodies to routine immunisations and commonly acquired infections, quantification of IgG subclasses

- Tests for phagocyte function: reduction of nitro blue tetrazolium chloride, assays of chemotaxis, bactericidal activity.

Due to the rarity of many primary immunodeficiencies, many of the above tests are highly specialised and tend to be performed in research laboratories.

Criteria for diagnosis were agreed in 1999. For instance, an antibody deficiency can be diagnosed in the presence of low immunoglobulins, recurrent infections and failure of the development of antibodies on exposure to antigens. The 1999 criteria also distinguish between "definitive", "probable" and "possible" in the diagnosis of primary immunodeficiency. "Definitive" diagnosis is made when it is likely that in 20 years, the patient has a >98% chance of the same diagnosis being made; this level of diagnosis is achievable with the detection of a genetic mutation or very specific circumstantial abnormalities. "Probable" diagnosis is made when no genetic diagnosis can be made, but the patient has all other characteristics of a particular disease; the chance of the same diagnosis being made 20 years later is estimated to be 85-97%. Finally, a "possible" diagnosis is made when the patient has only some of the characteristics of a disease are present, but not all.

The diagnosis of haemophilia C (factor XI deficiency) is centered on prolonged activated partial thromboplastin time.One will find that the factor XI has decreased in the individuals body.In terms of differential diagnosis one must consider:factor VIII deficiency, lupus anticoagulant and heparin contamination.